Biomedical Engineering Reference
In-Depth Information
recent developments in molecular biology have helped to provide mechanistic information on toxic-
ity [211]. The use of
in vitro
toxicity methods for the assessment of NPs was a subsequent devel-
opment and the applicability of a prevalent
in vitro
test to NPs is reviewed below. Sutter [212]
identified six general applications of
in vitro
toxicity assays, among which are the selection of
the most appropriate animal model of humans and the rapid screening of a series of toxicants. In
comparison to animal models,
in vitro
assays allow for a simpler, faster, and more cost-efficient
assessment of defined toxicity endpoints [213]. However,
in vitro
test systems lack the complexity
of animal models or the human body [211], and the metabolic activity of standardized cell lines has
often not been comprehensively characterized [214]. According to Snodin [215],
in vitro
systems
have no value for the prediction of biodistribution and target organ toxicity for the applied chemical
and its metabolites. Besides these considerations, scientists have been striving to determine the cor-
relation between the results obtained from
in vitro
and
in vivo
toxicity assessments. In the domain
of particle toxicology, for example, Sayes et al. [169] found little correlation between
in vitro
and
in vivo
pulmonary toxicity of several fine NPs. On the other hand, Donaldson et al. [98] reported
that the threshold for inflammation onset was identical
in vitro
and
in vivo
when the “particle sur-
face area burden per unit of proximal alveolar region surface area” of different low-toxicity, low-
solubility particles was used as a reference. Contradictory
in vitro
results on the same test substance
accumulating in the literature as well as the continuous public concern for animal welfare and safe
handling of substances have driven diverse international efforts to standardize and validate
in vitro
tests. The Economic Corporation & Development (OECD) validation harmonization report and
programs such as the MEIC (multicenter evaluation of
in vitro
toxicity, e.g., Ref. [216]), the German
Center for the Documentation and Validation of Alternative Methods (ZEBET), and standard proto-
cols made available via the INVITTOX database run by the European Centre for the Validation of
Alternative Methods (ECVAM), for example, are important aspects of an international standardiza-
tion and validation process (reviewed, e.g., in Ref. [217]).
In vitro
assays have general limitations for reliable risk assessment in the aspects of validation by
in vivo
experiments and a lack in standardized testing procedures, which are verified by reference
materials or interlaboratory validation.
19.13 CHALLENGES FOR NPs
IN VITRO
TEST METHODS
Physicochemical properties of NPs often limit the use of established
in vitro
toxicity assays for risk
assessment. Studies designed to determine NP toxicity should be ideally carried out using test sys-
tems that cannot be influenced by nanospecific properties [99]. Currently, however, NP risk analysis
is impaired by the lack of standardized test systems that fulfill these criteria.
New test systems for toxicity screening address new endpoints or new toxicity biomarkers. For
instance, cell culture systems reflecting better
in vivo
toxicity parameters have been recently devel-
oped [218]. New subdisciplines of toxicology, such as toxicogenomics, focus on studies of cellular
products controlled by the genome (RNA, proteins, and metabolites) and provide new approaches to
assess adverse biological effects of exogenous agents [219]. These technologies will enable a deeper
understanding of biochemical pathways and cellular responses.
However, many of these strategies depend on conventional detection systems and may still
be influenced by NP-specific properties. Novel technologies allowing marker-free cytotoxicity
testing might overcome these obstacles. For example, cellular analysis can be performed using
physical cell properties such as electrical resistance or refractive index. Digital holographic
microscopy, for example, detects the integral refractive index of living single cells in cell culture
medium, resulting in a phase shift of visible light [220]. Cell morphology reconstruction of the
digitally captured holograms is performed by application of a spatial phase shifting, a nondif-
fractive reconstruction method [221]. This method can be applied to a marker-free online analysis
of processes induced by drugs or toxic agents that lead to altered cell morphology, including
apoptosis and cell swelling [222]. Electrochemical impedance spectroscopy measures cellular
